Gene activation using FLP recombinase in C. elegans

The FLP enzyme catalyzes recombination between specific target sequences in DNA. Here we use FLP to temporally and spatially control gene expression in the nematode C. elegans. Transcription is blocked by the presence of an "off cassette" between the promoter and the coding region of the d...

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Bibliographic Details
Published in:PLoS genetics 2008-03, Vol.4 (3), p.e1000028
Main Authors: Davis, M Wayne, Morton, J Jason, Carroll, Dana, Jorgensen, Erik M
Format: Article
Language:English
Subjects:
Anatomy & physiology
Animals
Animals, Genetically Modified
Base Sequence
Biotechnology
Caenorhabditis elegans - enzymology
Caenorhabditis elegans - genetics
Cloning
DNA Nucleotidyltransferases - genetics
DNA Nucleotidyltransferases - metabolism
DNA Primers - genetics
Gene Expression Regulation
Genes, Helminth
Genetic engineering
Green Fluorescent Proteins - genetics
Heat
Heat-Shock Response - genetics
Luminescent Proteins - genetics
Medical research
Molecular Biology/Recombination
Neuroscience
Promoter Regions, Genetic
Proteins
Recombinant Fusion Proteins - genetics
Recombination, Genetic
Red Fluorescent Protein
Studies
Tetanus
Tetanus Toxin - genetics
Transcriptional Activation
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Summary:The FLP enzyme catalyzes recombination between specific target sequences in DNA. Here we use FLP to temporally and spatially control gene expression in the nematode C. elegans. Transcription is blocked by the presence of an "off cassette" between the promoter and the coding region of the desired product. The "off cassette" is composed of a transcriptional terminator flanked by FLP recognition targets (FRT). This sequence can be excised by FLP recombinase to bring together the promoter and the coding region. We have introduced two fluorescent reporters into the system: a red reporter for promoter activity prior to FLP expression and a green reporter for expression of the gene of interest after FLP expression. The constructs are designed using the multisite Gateway system, so that promoters and coding regions can be quickly mixed and matched. We demonstrate that heat-shock-driven FLP recombinase adds temporal control on top of tissue specific expression provided by the transgene promoter. In addition, the temporal switch is permanent, rather than acute, as is usually the case for heat-shock driven transgenes. Finally, FLP expression can be driven by a tissue specific promoter to provide expression in a subset of cells that can only be addressed as the intersection of two available promoters. As a test of the system, we have driven the light chain of tetanus toxin, a protease that cleaves the synaptic vesicle protein synaptobrevin. We show that we can use this to inactivate synaptic transmission in all neurons or a subset of neurons in a FLP-dependent manner.
ISSN:1553-7404
1553-7390
1553-7404
DOI:10.1371/journal.pgen.1000028